Abstract

1761

Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. P21 is an inhibitor of CDK4 and is a downstream transcription factor tightly regulated by p53.ATO is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (see poster Akay et al.). We hypothesized that knocking out of p53 or p21 will result in converting myeloma cells expressing w.t. p53 to behave like myeloma cell expressing mutant p53 with respect to activating the extrinsic apoptotic pathway. Thus, IM9 cells were transfected (AMAXA Nucleoporator) with a GFP-SiRNA expressing plasmid co-expressing anti-sense sequences for p53 or p21 (InvivoGen). Cells were also transfected with the same vector with no SiRNA (empty vector). Transfection rate was 30-50%, by GFP (flow cytometry). We observed in control cells (empty vector) a G1 arrest through activation of p21; release of cytochrome C to the cytosol and apoptosis. In cells transfected with the p53SiRNA plasmid or the p21SiRNA plasmid, we observed primarily activation of the extrinsic apoptotic pathway with activation of caspase 8 and BID; down regulation of FLIP and release of AIF from mitochondria to the cytosol. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer types.